Methods of using pvt1 exon 9 to diagnose and treat prostate cancer

ABSTRACT

PVT1 exon 9 is overexpressed in aggressively tumorigenic prostate cancer cell lines and prostate tumor tissues. This exon provides a diagnostic tool for the detection and monitoring of aggressive prostate cancer. Several small interfering ribonucleic acids (siRNAs) are disclosed that are useful for treating prostate cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/345,463 (filed Nov. 7, 2016) which claims priority to and is anon-provisional of U.S. Patent Applications 62/251,210 (filed Nov. 5,2015) and 62/300,961 (filed Feb. 28, 2015), the entirety of which areincorporated herein by reference.

STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under grant numbers G12MD007599 awarded by the National Institute on Minority Health and HealDisparities. The government has certain rights in the invention.

REFERENCE TO A SEQUENCE LISTING

This application refers to a “Sequence Listing” listed below, which isprovided as an electronic document entitled “Sequence.txt” (9 kb createdon Nov. 30, 2017) which is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

Prostate cancer (PCa) is the most common non-cutaneous cancer and thesecond leading cause of cancer-related death for men in the U.S. It isestimated that, in 2015, approximately 220,800 new cases of PCa will bediagnosed and 27,540 deaths will result from PCa. African Americans havethe highest incidence of PCa in the world, with an annual average of 229per 100,000 men for the period of 2006-2010, which represents abouttwo-fold more than Caucasian Americans. PCa is also the leading cancerin terms of incidence and mortality in men from Africa and theCaribbean. Consequently, African ancestry is a very important riskfactor.

PCa is a heterogeneous disease, with multiple risks factors. Thespecific reasons for poor outcomes from PCa in males of African ancestry(MoAA) when compared to Caucasian males (CM) are not understood.However, it is widely believed that the causes of PCa disparities arecomplex and multifaceted. Two potential reasons are frequently proposedto explain this profound disparity in PCa: (1) MoAA present more oftenthan CM with advanced incurable PCa due to more limited access to healthcare; (2) PCa is biologically more aggressive in MoAA than CM, and canbe attributed to environmental and/or genetic risk factors.

The 8q24 human chromosomal region is one of the most importantsusceptibility genetic loci for PCa. Several studies have identifiedsingle nucleotide polymorphisms (SNPs) located in chromosome 8q24 assusceptibility markers for PCa. The 8q24 chromosomal region has only oneprotein-coding gene, the well-known MYC oncogene implicated in differentcancers, including PCa. However, it also has a number of non-proteincoding genes (such as PVT1) whose functional roles have not beenthoroughly investigated yet.

In recent years, non-protein coding RNAs (ncRNAs) have received specialattention because they have been identified in many studies as beingimportant in cancer biology. Substantial progress has been made inunderstanding the role of small non-coding RNAs such as microRNAs(miRNAs) in the development and progression of cancers. However,studying the role of long non-coding RNAs (lncRNAs) in cancers appearsto be more complicated. LncRNAs are defined as endogenous cellular RNAsthat have a size of more than 200 nucleotides and that do not possess anextended open reading frame.

The discussion above is merely provided for general backgroundinformation and is not intended to be used as an aid in determining thescope of the claimed subject matter.

BRIEF DESCRIPTION OF THE INVENTION

PVT1 exon 9 is overexpressed in aggressively tumorigenic prostate cancercell lines and prostate tumor tissues. This exon provides a diagnostictool for the detection and monitoring of aggressive prostate cancer.Several small interfering ribonucleic acids (siRNAs) are disclosed thatare useful for treating prostate cancer.

This brief description of the invention is intended only to provide abrief overview of subject matter disclosed herein according to one ormore illustrative embodiments, and does not serve as a guide tointerpreting the claims or to define or limit the scope of theinvention, which is defined only by the appended claims. This briefdescription is provided to introduce an illustrative selection ofconcepts in a simplified form that are further described below in thedetailed description. This brief description is not intended to identifykey features or essential features of the claimed subject matter, nor isit intended to be used as an aid in determining the scope of the claimedsubject matter. The claimed subject matter is not limited toimplementations that solve any or all disadvantages noted in thebackground.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the manner in which the features of the invention can beunderstood, a detailed description of the invention may be had byreference to certain embodiments, some of which are illustrated in theaccompanying drawings. It is to be noted, however, that the drawingsillustrate only certain embodiments of this invention and are thereforenot to be considered limiting of its scope, for the scope of theinvention encompasses other equally effective embodiments. The drawingsare not necessarily to scale, emphasis generally being placed uponillustrating the features of certain embodiments of the invention. Inthe drawings, like numerals are used to indicate like parts throughoutthe various views. Thus, for further understanding of the invention,reference can be made to the following detailed description, read inconnection with the drawings in which:

FIG. 1 is schematic depiction of the PCa susceptibility locus;

FIG. 2A, FIG. 2B and FIG. 2C are graphs depicting expression of PVT1exons 1a; 1b and 1c in non-tumorigenic and tumorigenic prostateepithelial cell lines;

FIG. 3A, FIG. 3B and FIG. 3C are graphs depicting expression of PVT1exons 2; 3b and 4a in non-tumorigenic and tumorigenic prostateepithelial cell lines;

FIG. 4A to FIG. 4C are graphs depicting expression of PVT1 exons 4b, 5,6 (FIG. 4A, FIG. 4B and FIG. 4C, respectively);

FIG. 5A and FIG. 5B are graphs depicting expression of exons 7 and 8,respectively;

FIG. 6 is a graph depicting PVT1 exon 9 overexpression in aggressivelytumorigenic PCa cell line from males of African ancestry;

FIG. 7 is a graph depicting PVT1 exon 9 expression in non-tumorigenicand tumorigenic prostate epithelial cell lines;

FIG. 8 is a graph depicting expression of PVT1 exon 9 on select PCa celllines that are exposed to the five siRNAs;

FIG. 9 is a graph depicting the relative fold of apoptosis induced byselect siRNAs;

FIG. 10 is a graph showing the siRNAs arrest cell cycle progression atthe G1 phase of the cell cycle;

FIG. 11 is a graph depicting relative percentage of proliferative cellswhen exposed to a select siRNA;

FIG. 12 is a graph showing relative expression of PVT1 exon 4a invarious prostate tissues;

FIG. 13 is a graph showing relative expression of PVT1 exon 4b invarious prostate tissues; and

FIG. 14 is a graph showing relative expression of PVT1 exon 9 in variousprostate tissues;

DETAILED DESCRIPTION OF THE INVENTION

Prostate cancer has been shown to be associated with single nucleotidepolymorphisms around regions 2 and 3 of the 8q24 human chromosomalregion. The non-protein coding gene locus Plasmacytoma VariantTranslocation 1 (PVT1) is located at 8q24 and is overexpressed in PCaand, therefore, is also a candidate biomarker to explain the well-knowndisparity among males of African ancestry. PVT1 has at least twelveexons that make separate transcripts which may have different functions,all of which are, at present unknown, in PCa. The disclosed studydetermined if any PVT1 transcripts play a role in aggressiveness andracial disparity in PCa. A panel of seven PCa cell lines was used,including three derived from males of African ancestry. Ribonucleic acidextraction, complementary deoxyribonucleic acid synthesis, andquantitative polymerase chain reaction (qPCR) were performed to evaluateexpression of all twelve PVT1 exons. Each qPCR was performed inquadruplicates. At least four separate qPCR experiments were performed.Expression of PVT1 exons was inconsistent except for exon 9. There wasno significant difference in exon 9 expression between cell linesderived from Caucasian males (CM), and an indolent cell line derivedfrom males of African ancestry. However, exon 9 expression in theaggressive MDA PCa 2b and E006AA-hT cell lines derived from males ofAfrican ancestry was significantly higher than in other cell lines.Consequently, differential expression of exon 9 of PVT1 was observed ina manner that suggests that PVT1 exon 9 may be associated withaggressive PCa in males of African ancestry.

FIG. 1 depicts the PCa susceptibility locus. The PVT1 gene locusexpresses several alternatively spliced non-protein coding transcriptsand it also encodes a cluster of microRNAs. However, no specificfunctional role for any of these transcripts has been identified. PVT1is located downstream of MYC. It has a size of over 300 kb and, sinceits discovery in the mid-80s, it has been proven to play an importantrole in cancer. The upregulation of PVT1 has been found to be involvedin poor prognosis in colorectal cancer and gastric cancer. In non-smallcell lung cancer, it promotes tumorigenesis. In PCa, PVT1 has been foundto have an increased expression in comparison to normal prostate tissuewith the presence of a newly identified functional PCa specific geneticvariant, rs378854. PVT1 encodes for several transcripts, approximatelytwelve exons, and their differential expression has not previously beeninvestigated. Consequently, in the present disclosure data on therelative expression of the twelve different exons of PVT1 in modelsrepresentative of a wide variety of clinical PCa. Observations indicatethat exon 9 of PVT1 is significantly overexpressed in PCa derived fromaggressive PCa in MoAA, thus suggesting the potential for clinicalutility in this population group least served by current managementstrategies.

Cell Lines

For this study, seven prostate epithelial cell lines were used, inrepresentation of the heterogeneity notable in clinical PCa. The RWPE1cells are epithelial cells derived from the peripheral zone of ahistologically normal prostate from a 54-year-old CM. The cells werelater transfected with a single copy of the human papilloma virus 18(HPV-18) to finally establish the cell line. RWPE1 is non-tumorigenic.WPE1-NA22 cells were obtained by exposing RWPE1 toN-methyl-N-nitrosourea. WPE1-NA22 cells are mildly tumorigenic. MDA PCa2b cells were derived from bone metastasis of prostate adenocarcinoma ina 64-year-old MoAA. PC-3 was derived from bone metastasis of a grade IVprostatic adenocarcinoma from a 62-year-old CM. The VCaP cell line wasestablished from a vertebral bone metastasis from a 59-year-old CM withhormone refractory PCa. The E006AA cell line was established from a50-year-old MoAA who underwent radical retropubic prostatectomy fortreatment of clinically-localized PCa. The E006AA cell line isnon-tumorigenic in nude mice. The highly tumorigenic derivative ofE006AA, the E006AA-hT cell line, was established and characterized in2014. The main features of all the cell lines used in this study aresummarized in Table 1.

All the cell lines were purchased from the American Type CultureCollection (ATCC, Manassas, Va., USA) except for the E006AA andE006AA-hT cell lines that were a kind gift from Dr. Shariar Koochekpourof the Roswell Park Cancer Institute (New York, USA).

TABLE 1 Main characteristics of prostate epithelial cell lines used inthis study Aggres- Indolent sive Normal Androgen Cell line CM MoAADisease Disease Prostate Status RWPE1 X X Dependent WPE1-NA22 X XDependent MDA X X Dependent PCa 2b E006AA X X Independent E0006AA- X XIndependent hT PC-2 X X Independent VCaP X X Dependent

Cell Culture and Cell Culture Reagents

RWPE1 and WPE1-NA22 cells were maintained in Keratinocyte-Serum FreeMedium (Life Technologies, Grand Island, N.Y., USA) supplemented with0.05 mg/mL bovine pituitary extract and 5 ng/mL human Epidermal GrowthFactor. MDA PCa 2b cells were maintained in F-12K medium supplementedwith 20% fetal bovine serum (FBS), 25 ng/mL cholera toxin, 10 ng/mLmouse epidermal growth factor, 0.005 mM phosphoethanolamine, 100 pg/mLhydrocortisone, 45 nM selenous acid and 0.005 mg/mL bovine insulin. PC-3cells were maintained in F-12K medium supplemented with 10% FBS. VCaPcells were maintained in DMEM medium supplemented with 10% FBS. E006AAand E006AA-hT cells were maintained in DMEM medium supplemented with 10%FBS. All cell lines were also cultured with the presence of 1%Penicillin/Streptomycin.

Primer Design and Sequences

The following PVT1 exons were previously described: 1a, 2, 3b, 4b, 7, 8and 9 (Frontiers in Genetics, 20, Apr. 2012, vol 3, article 69).However, using the UCSC Genome browser, the PVT1 sequence was carefullyannotated and twelve exons were retrieved from the analysis. ThePrimers3 Plus software was used to custom-design primers for all twelveannotated exons, and the sequences of the primers are listed in Table 2.

TABLE 2  List of primer sequences of PVT1 exons Primer NamePrimer Sequence 5′-3′ PVT1 Exon 1A-F ACGAGCTGCGAGCAAAGA (SEQ ID NO: 1)PVT1 Exon 1A-R CGTGTCTCCACAGGTCACAG (SEQ ID NO: 2) PVT1 Exon 1B-FCGGAAGCTGCAGAAGGACAAA (SEQ ID NO: 3) PVT1 Exon 1B-RCTCAAATAATGGAGACCAGGCCA  (SEQ ID NO: 4) PVT1 Exon 1C-FGCAGTGCAGGAAGCCAACTA (SEQ ID NO: 5) PVT1 Exon 1C-RCTTAGGGGTCCTTACAGCCAAG  (SEQ ID NO: 6) PVT1 Exon 2-FAACCATGCACTGGAATGACA (SEQ ID NO: 7) PVT1 Exon 2-RCATCAGATGCTTCACCAGGA (SEQ ID NO: 8) PVT1 Exon 3B-FCATACTCCCTGGAGCCTTCTC (SEQ ID NO: 9) PVT1 Exon 3B-RCAGTGTCCTGGCAGTAAAAGG  (SEQ ID NO: 10) PVT1 Exon 4A-FGGGTTCAAGTGATCCTCCTG (SEQ ID NO: 11) PVT1 Exon 4A-RTGTAATCCCAGCACGTTGAA (SEQ ID NO: 12) PVT1 Exon 4B-FCACCTGGGATTTAGGCACTT (SEQ ID NO: 13) PVT1 Exon 4B-RCCAATCTCAAAATACTCCAGCTTT  (SEQ ID NO: 14) PVT1 Exon 5-FGCCAACAGAGATTTTGAGAAACAC  (SEQ ID NO: 15) PVT1 Exon 5-RTCAGCTCAGGTTCCCATTGT (SEQ ID NO: 16) PVT1 Exon 6-FTGCTAGGGTGACAGAAACTGG  (SEQ ID NO: 17) PVT1 Exon 6-RCCCAGGTCTTGATGACAGGT (SEQ ID NO: 18) PVT1 Exon 7-FTTGGTGCTCTGTGTTCACCT (SEQ ID NO: 19) PVT1 Exon 7-RTGTCCACTAGCAGCAACAGG (SEQ ID NO: 20) PVT1 Exon 8-FAGAATAACGGGCTCCCAGAT (SEQ ID NO: 21) PVT1 Exon 8-RAAGCTGGGTCTTCATCCTGA (SEQ ID NO: 22) PVT1 Exon 9-FCATGACTCCACCTGGACCTT (SEQ ID NO: 23) PVT1 Exon 9-RGTGGGCGATGAAGTTCGTA (SEQ ID NO: 24)

RNA Extraction and RT-QPCR

At 75% confluency, total RNA was extracted from cells in a 60×15 mmtissue culture dish, using RNeasy Mini Kit (Qiagen, Germany, cat#74104).After quantification with Nanodrop1000 spectrophotometer (NanoDrop,Madison, Wis., USA), 1 μg of RNA was reverse-transcribed into cDNA usingQuantiTect Reverse Transcription kit (Qiagen, Germany, cat#205311).Amplification reactions were performed in 25 reaction volume using SYBRGreen PCR master Mix (Life Technologies, Grand Island, N.Y., USAcat#4309155), cDNA template and 0.4 μM final concentration for primers.The thermal cycle profile employed was as follows: 50° C. for 2 min, 10min initial denaturation at 95° C., and 40 cycles of 15 s denaturationat 94° C., 1 min annealing at 65° C. A dissociation curve was also addedat the end of the cycle. The amplifications were carried out on the 7500Real Time PCR machine (Applied Biosystems instruments, Grand Island,N.Y., USA). Messenger RNA (mRNA) expression was assessed inquadruplicates in at least 3 independent experiments and normalized toRPL32 mRNA expression. Relative expression levels were calculated by theCt method (ΔΔ Ct). Previously published RPL32 primer sequences wereused.

Statistical Analysis

Data are presented as mean±standard error of the mean (S.E.M) of atleast three independent experiments. Statistical significance ofdifferences was assessed using two-tailed Student's t test. p valuesless than 0.05 were deemed significant. A summary of all p valuesresulting from comparing each prostate cell line to the normal prostatecell line RWPE1 for all PVT1 exons are summarized in Table 3.

TABLE 3 Summary of p values comparing each prostate cell line to thenormal prostate cell line RWPE1 for all PVT1 exons WPE1- MDA E006AA-Caption NA22 PCa 2b E006AA hT PC-3 VCaP Exon 1A 0.4148 0.1017 0.28650.0262 Exon 1B 0.2950 0.0133 0.0060 0.3158 Exon 1C 0.2864 0.2326 0.06860.0369 Exon 2 0.3284 0.0016 0.0114 0.2598 Exon 3B 0.4073 0.4516 0.03590.1179 Exon 4A 0.4410 0.0010 0.3320 0.2397 0.1518 0.0633 Exon 4B 0.07800.4943 0.0130 0.4064 Exon 5 0.1867 0.0244 0.3641 0.0938 Exon 6 0.11600.4997 0.3313 0.0409 Exon 7 0.1976 0.1799 0.3066 0.1383 Exon 8 0.14920.2963 0.0255 0.0650 Exon 9 0.0179 0.0050 0.0742 0.0416 0.0007 0.3552

For PVT1 exon 1a and 1c, no significant differential expression wasobserved in the MDA PCa 2b PCA cell line derived from a male of Africanancestry, when compared to the RWPE1 normal prostate epithelial cellline. For PVT1 exon 1b, a decrease in the expression level was observedin the MDA PCa 2b cell line, but it was not significant. For PVT1 exon2, a small but significant decrease (p value=0.00161) in relativeexpression by MDA PCa 2b was observed in comparison to RWPE1. However,for PVT1 exon 3b, no difference in relative expression was observed whenMDA PCa 2b was compared to RWPE1. For PVT1 exon 4a, a significantdecrease in relative expression by MDA PCa 2b of almost 60% incomparison to RWPE1 was observed (p value=0.001104). However, PVT1 Exon4a was overexpressed in the highly tumorigenic E006AA-hT also derivedfrom a MoAA. However, this overexpression was not statisticallysignificant (p value=0.2397). Given that both MDA PCa 2b and E006AA-hTare highly tumorigenic and derived from MoAA, the dissimilar expressionof PVT1 exon 4a in them suggests inconsistency. The reasons for this arecurrently unclear.

FIG. 2A, FIG. 2B and FIG. 2C depict expression of PVT1 exons 1a; 1b and1c in non-tumorigenic and tumorigenic prostate epithelial cell lines.Four independent qPCR experiments were performed and every experimentwas set up in quadruplicates. The data showed that there is nosignificant differential expression of the exons in the cell lines incomparing cell lines derived from CM with those derived from MoAA. Thedata are presented as mean+standard error of the mean (SEM).

FIG. 3A, FIG. 3B and FIG. 3C depict PVT1 exons 2; 3b and 4a expressionin non-tumorigenic and tumorigenic prostate epithelial cell lines. Four(for exon 2 and exon 3b) or 3 (for exon 4a) independent qPCR experimentswere performed and every experiment was set up in quadruplicates. Thedata showed that there is no significant differential expression of theexons in the cell lines in comparing cell lines derived from CM withthose derived from MoAA. The data are presented as mean±standard errorof the mean (SEM).

FIG. 4A to FIG. 5B depict expression of PVT1 exons 4b, 5, 6 (FIG. 4A,FIG. 4B and FIG. 4C) and exons 7 and 8 (FIG. 5A and FIG. 5B). There wereno differential expression observed in comparing MDA PCa 2b to RWPE1.PVT1 exons 4b (FIG. 4A); 5 (FIG. 4B) and 6 (FIG. 4C) expression innon-tumorigenic and tumorigenic prostate epithelial cell lines. Fourindependent qPCR experiments were performed and every experiment was setup in quadruplicates. The data showed that there was no significantdifferential expression of the exons in the cell lines in comparing celllines derived from CM with those derived from MoAA. The data arepresented as mean+standard error of the mean (SEM). PVT1 exons 7 (FIG.5A) and 8 (FIG. 5B) expression in non-tumorigenic and tumorigenicprostate epithelial cell lines. Four independent qPCR experiments wereperformed and every experiment was set up in quadruplicates. The datashowed that there is no significant differential expression of the exonsin the cell lines in comparing cell lines derived from CM with thosederived from MoAA. The data are presented as mean+standard error of themean (SEM).

FIG. 6 depicts PVT1 exon 9 overexpression in aggressively tumorigenicPCa cell line from MoAA. Five independent qPCR experiments wereperformed and every experiment was set up in quadruplicates. The datashowed that PVT1 exon 9 is consistently significantly overexpressed inthe aggressive PCa cell lines derived from MoAA in comparison to thenon-tumorigenic PCa cell line it was derived from. The data arepresented as mean+standard error of the mean (SEM) and the p values aredisplayed on FIG. 6. The lower panel shows the different prostate celllines' PVT1 exon 9 PCR products loaded on a 0.8% agarose gel.

PVT1 exon 9 was the only one of all twelve exons of PVT1 that showed avery consistent and easily explainable expression profile. PVT1 exon 9was significantly and consistently overexpressed in both aggressivelytumorigenic cell lines derived from men of African ancestry. In both MDAPCa 2b and E006AA-hT cell lines, relative expression of PVT1 exon 9 incomparison to the RWPE1 cell line were approximately 200% (two-fold)higher (FIG. 6). Interestingly, in comparing the E006AA non-tumorigeniccell line derived from a MoAA with its derivative, the aggressivelytumorigenic E006AA-hT cell line, PVT1 exon 9 expression was about 300%higher in the E006AA-hT cell line (p=0.0487; FIG. 7). This indicatesthat PVT1 exon 9 is related to aggressiveness in this model of PCa in aMoAA.

FIG. 7 depicts PVT1 exon 9 expression in non-tumorigenic and tumorigenicprostate epithelial cell lines. At least three independent qPCRexperiments were performed and every experiment was set up inquadruplicates. The data showed that PVT1 exon 9 is consistentlysignificantly overexpressed in the aggressive PCa cell lines derivedfrom MoAA. The data are presented as mean±standard error of the mean(SEM) and the p values are displayed on FIG. 7, and are compared to thecontrol (RWPE1 cell line).

The long non-protein coding RNA (lncRNA) PVT1 has been shown to beimportant in cancer. PVT1 overexpression has been demonstrated inpancreatic cancer and colon cancer, and is related to poor prognosis inmost of these cases. However, these studies did not distinguish betweenthe different transcripts of PVT1. It is possible that the differentexons of PVT1 could be differentially expressed, and have differentfunctions. Here, a study was performed where primers were annotated anddesigned for amplification of twelve separate exons of PVT1. The resultsshow that most of the exons do not have differential expression in PCa.However, very interestingly, one particular exon of PVT1, PVT1 exon 9,was consistently and significantly overexpressed in the aggressive PCacell lines derived from MoAA. Importantly, PVT1 exon 9 is demonstratedto be significantly overexpressed in the aggressive PCa cell linederived from a MoAA in comparison to its non-tumorigenic cell line fromwhich it was derived. This clearly indicates that PVT1 exon 9overexpression is significantly associated with aggressiveness in thismodel of PCa in a MoAA. Whether this will translate to individuals orsubpopulations at risk prospectively is at present unknown. A more indepth evaluation of prospectively collected PCa gene databases and freshprostate specimens will be required to understand whether thisphenomenon can be used as a biomarker for MoAA with PCa. Already,emerging data from an ongoing clinical study strongly indicates thatPVT1 exon 9 is overexpressed in prostate tumor tissue in comparison tobenign prostate tissue, and even much more so in comparison to normalprostate tissue.

The physical characteristics of the custom-designed PVT1 exon 9 primersare as follows: the disclosed PVT1 exon 9 primers amplify a region of210 bp in the PVT1 exon 9 sequence. In order to design PVT1 exon 9primers, the PVT1 exon 9 sequence shown below was used.

The PVT1 exon 9 sequence is given by:GTTTTTTGCATGTCTGACACCCATGACTCCACCTGGACCTTATGGCTCCACCCAGAAGCAATTCAGCCCAACAGGAGGACAGCTTCAACCCATTACGATTTCATCTCTGCCCCAACCACTCAGCAGCAAGCACCTGTTACCTGTCCACCCCCACCCCTTCCCCCAAACTGCCTTTGAAAAATCCCTAACCTATGAGCTTTGAATAAGATGAGTACGAACTTCATCGCCCACGTGGCGTGGCCGGCCTCGTGTCTATTAA ATTCTTTTTCTACT (SEQID NO: 25)

The corresponding forward and reverse primers designed are given as SEQID NO: 23 and SEQ ID NO: 24.

To determine if a lncRNA is important, its cellular functions need to beelucidated. It will be important to determine if the lncRNA regulatesimportant cellular functions or if it just represents “transcriptionalnoise” or background transcription. Although lncRNAs are sometimesaberrantly expressed in diseased tissues, suggesting specific functionsin diseases, knowledge of how these lncRNA act in the cell and whichroles they might play in diseases in still very limited. Therefore, anunderstanding of the cellular functions, mechanisms of action, andmechanisms regulating PVT1 exon 9 expression will be critical toexploiting it for potential clinical applications.

siRNAs as Inhibitors

Five different siRNAs were custom-designed specifically targeting PVT1exon 9. As PVT1 exon 9 had not been previously described in literature,there were no siRNAs to PVT1 exon 9 available. These five newcustom-designed PVT1 exon 9 siRNAs are very valuable tools that are veryuseful in understanding the function of PVT1 exon 9 in prostate cancer.Data show that these siRNAs may have therapeutic application in prostatecancer. The sequences and main characteristics of all duplexes are shownin Table 4A and Table 4B.

TABLE 4A  Oligo Name Sequence (5′-3′) PVT1 exon9_siRNA 1FCUUCAACCCAUUACGAUUUUU  (SEQ ID NO: 26) PVT1 exon9_siRNA 1RAAAUCGUAAUGGGUUGAAGUU  (SEQ ID NO: 27) PVT1 exon9_siRNA 2FGGACAGCUUCAACCCAUUAUU  (SEQ ID NO: 28) PVT1 exon9_siRNA 2RUAAUGGGUUGAAGCUGUCCUU  (SEQ ID NO: 29) PVT1 exon9_siRNA 3FAGGACAGCUUCAACCCAUUUU  (SEQ ID NO: 30) PVT1 exon9_siRNA 3RAAUGGGUUGAAGCUGUCCUUU  (SEQ ID NO: 31) PVT1 exon9_siRNA 4FCCAUUACGAUUUCAUCUCUUU  (SEQ ID NO: 32) PVT1 exon9_siRNA 4RAGAGAUGAAAUCGUAAUGGUU  (SEQ ID NO: 33) PVT1 exon9_siRNA 5FACCUAUGAGCUUUGAAUAAUU  (SEQ ID NO: 34) PVT1 exon9_siRNA 5RUUAUUCAAAGCUCAUAGGUUU  (SEQ ID NO: 35)

TABLE 4B Secondary Oligo Name Length m.w. Tm Dimers structure GC % PVT1exon9_siRNA 1F 21 13278 50.5 No None 33.3 PVT1 exon9_siRNA 1R 21 1327850.5 No None 33.3 PVT1 exon9_siRNA 2F 21 13308 58.1 No None 42.8 PVT1exon9_siRNA 2R 21 13308 58.1 No Very weak 42.8 PVT1 exon9_siRNA 3F 2113308 59.5 No None 42.8 PVT1 exon9_siRNA 3R 21 13308 59.5 No Very weak42.8 PVT1 exon9_siRNA 4F 21 13278 50.1 No Very weak 33.3 PVT1exon9_siRNA 4R 21 13278 50.1 No Very weak 33.3 PVT1 exon9_siRNA 5F 2113263 49.9 Yes None 28.5 PVT1 exon9_siRNA 5R 21 13263 49.9 No None 28.5

In one embodiment, the sequences disclosed in Table 4A comprise anoverhang on their 3′ end.

The immediate application PVT1 exon 9 siRNAs is the silencing of PVT1exon 9 endogenous expression in cells in order to study the effects ofloss of expression of PVT1 exon 9 on various cellular functions. Thedata indicate that these PVT1 exon 9 siRNAs may have therapeuticpotential in aggressive prostate cancer. Silencing of PVT1 exon 9expression using these siRNAs resulted in induction of apoptosis, cellcycle arrest at G1 phase, dephosphorylation of the retinoblastomaprotein, and loss of expression of the proliferating cell nuclearantigen (PCNA) protein.

FIG. 8 depicts expression of PVT1 exon 9 (relative to RPL32) on selectPCa cell lines that are exposed to the five siRNAs. Cells weretransfected with 30 pM siRNA for 72 hours. FIG. 8 shows the response ofMDA PCa 2b, E006AA and E006AA-hT cell lines exposed to siRNA 1, siRNA 2,siRNA3, siRNA4 or siRNA5. A control (siScrbl) scrambled sequence is alsoprovided.

FIG. 9 is a graph depicting the relative fold of apoptosis (as a percentof control) induced by select siRNA5. The data indicates a two-foldincrease in apoptosis when 30 pM siRNA5 is utilized.

FIG. 10 is a graph showing the effect of 30 pM siRNA5 in inducing arrestof E006AA-hT PCa cells at the G1 phase of cell cycle.

FIG. 11 is a graph showing the relative percentage of proliferativecells after treatment with 30 pM siRNA5 on the E006AA-hT PCa cell lineafter 72 hours;

FIG. 12 is a graph showing relative expression of PVT1 exon 4a of sevenbenign prostate tissues; seven prostate tumor tissues and seven normalprostate tissues. Total RNA were extracted from human prostate tissue;seven samples of normal prostate tissue (NT), seven samples from benignprostate tissue and seven samples of prostate tumor tissues. Synthesisof cDNA were performed with 450 ng of RNA.

FIG. 13 is a graph showing relative expression of PVT1 exon 4b in sevenbenign prostate tissues; seven prostate tumor tissues and seven normalprostate tissues.

FIG. 14 is a graph showing relative expression of PVT1 exon 9 in sevenbenign prostate tissues; seven prostate tumor tissues and seven normalprostate tissues.

In one embodiment, a small interfering ribonucleic acid (siRNA) isprovided that comprises a double stranded siRNA with fewer thantwenty-six nucleotides. The double stranded siRNA comprises a firststrand comprising a primary structure and a first 3′ overhang, theprimary structure being selected from the group consisting ofCUUCAACCCAUUACGAUUU (SEQ ID NO. 26); GGACAGCUUCAACCCAUUA (SEQ ID NO.28); AGGACAGCUUCAACCCAUU (SEQ ID NO. 30); CCAUUACGAUUUCAUCUCU (SEQ IDNO. 32); and ACCUAUGAGCUUUGAAUAA (SEQ ID NO. 34). The siRNA furthercomprises a second strand that is complementary to the first strand, thesecond strand having a second 3′ overhang. In one embodiment, the first3′ overhang is UU and the second 3′ overhang is UU. In anotherembodiment, the double stranded siRNA has twenty-one nucleotides. Inanother embodiment, the first 3′ overhang is UU and the second 3′overhang is UU and the double stranded siRNA has twenty-one nucleotides.

In another embodiment a method of treating aggressive prostate cancer isdisclosed. The method comprises administering to a prostate tumor asmall interfering ribonucleic acid (siRNA) comprising a double strandedsiRNA with fewer than twenty-six nucleotides, the double stranded siRNAcomprising: a first strand comprising a primary structure and a first 3′overhang, the primary structure being selected from the group consistingof CUUCAACCCAUUACGAUUU (SEQ ID NO. 26); GGACAGCUUCAACCCAUUA (SEQ ID NO.28); AGGACAGCUUCAACCCAUU (SEQ ID NO. 30); CCAUUACGAUUUCAUCUCU (SEQ IDNO. 32); and ACCUAUGAGCUUUGAAUAA (SEQ ID NO. 34). The siRNA comprises asecond strand that is complementary to the first strand, the secondstrand having a second 3′ overhang.

The PVT1 exon 4A sequence is given by:

(SEQ ID NO: 36) AGTCTCACTCTGTGGTCCAGGCTGAAGTACAGTGGCATGATCCCAGGTCACTGCAACCCCCACCTCCCGGGTTCAAGTGATCCTCCTGCCTCAGCCTCCCGAGTAGCTGGTATTACAGGCGTGTGCCACAAAGCCTGGCTAAGTTTTGTATTTTTAGTAGAGACGGGGTTTCACCATGTTGGCCAGGTTGGTCTCGAACTCCTGACCTCAAGTGATCCACTCACTTTGGCCTTTCAACGTGCTGGGATTACAGGCGAGAGTCACCGCACCCGGACGACTCTGACATTTTTGAAGAGTCCA G.

The PVT1 exon 4B sequence is given by:

(SEQ ID NO: 37) AATCCTGTTACACCTGGGATTTAGGCACTTTCAATCTGAAAAAATACATATCCTTTCAGCACTCTGGACGGACTTGAGAACTGTCCTTACGTGACCTAAAGCTGGAGTATTTTGAGATTGGAGAATTAAG

This written description uses examples to disclose the invention,including the best mode, and also to enable any person skilled in theart to practice the invention, including making and using any devices orsystems and performing any incorporated methods. The patentable scope ofthe invention is defined by the claims, and may include other examplesthat occur to those skilled in the art. Such other examples are intendedto be within the scope of the claims if they have structural elementsthat do not differ from the literal language of the claims, or if theyinclude equivalent structural elements with insubstantial differencesfrom the literal language of the claims.

SEQUENCE LISTING

<110> Research Foundation of the City University of New York

<120> METHODS OF USING PVT1 EXON 9 TO DIAGNOSE AND TREAT PROSTATE CANCER

<130> RESE.17663DIV-RO

<150> 62/251,210

<151> 2015-11-05

<150> 15/345,463

<151> 2016-11-07

<160> 37

What is claimed is:
 1. A method of producing an analyte, the methodcomprising sequential steps of: obtaining a sample of prostate tissue;extracting ribonucleic acid (RNA) from the prostate tissue; performingquantitative polymerase chain reaction (qPCR) on the RNA to amplify aPVT1 exon 9 sequence given by SEQ ID NO: 25, the qPCR generatingreplicated fragments that are useful as analytes.
 2. The method recitedin claim 1, further comprising steps of quantifying a concentration ofthe replicated fragments; comparing the concentration of the replicatedfragments to a predetermined threshold concentration, wherein theprostate tissue is deemed to be aggressive prostate cancer if theconcentration exceeds the predetermined threshold.
 3. The method asrecited in claim 2, wherein the step of performing quantitativepolymerase chain reaction (qPCR) utilizes a forward primer with asequence given by SEQ ID NO. 23 and a reverse primer with a sequencegiven by SEQ ID NO.
 24. 4. A method of producing an analyte, the methodcomprising sequential steps of: obtaining a sample of prostate tissue;extracting ribonucleic acid (RNA) from the prostate tissue; performingquantitative polymerase chain reaction (qPCR) on the RNA to amplify aPVT1 exon 4A sequence given by SEQ ID NO: 36 or a PVT1 exon 4B sequencegiven by SEQ ID NO. 37, the qPCR generating replicated fragments thatare useful as analytes.
 5. The method recited in claim 4, furthercomprising steps of quantifying a concentration of the replicatedfragments; comparing the concentration of the replicated fragments to apredetermined threshold concentration, wherein the prostate tissue isdeemed to be aggressive prostate cancer if the concentration exceeds thepredetermined threshold.
 6. The method as recited in claim 5, whereinthe step of performing quantitative polymerase chain reaction (qPCR)utilizes a forward primer with a sequence given by SEQ ID NO. 11 and areverse primer with a sequence given by SEQ ID NO.
 12. 7. The method asrecited in claim 5, wherein the step of performing quantitativepolymerase chain reaction (qPCR) utilizes a forward primer with asequence given by SEQ ID NO. 13 and a reverse primer with a sequencegiven by SEQ ID NO. 14.